ß 2007 Wiley-Liss, Inc. American Journal of Medical Genetics Part A 143A:2815–2820 (2007)

Clinical Report A Familial Case of Type II Caused by a Dominant COL2A1 Mutation and ‘‘Patchy’’ Expression in the Mosaic Father

F. Forzano,1* M. Lituania,2 V. Viassolo,1 A. Superti-Furga,3 G. Wildhardt,5 B. Zabel,3,4 and F. Faravelli1 1S.C. Genetica Umana, Ospedali Galliera, Genova, Italy 2S.S. Medicina Fetale, Ospedali Galliera, Genova, Italy 3Centre for Pediatrics, University of Freiburg, Freiburg, Germany 4Children’s Hospital, University of Mainz, Mainz, Germany 5Bioscientia, Center for Human Genetics, Ingelheim, Germany Received 20 July 2006; Accepted 5 August 2007

Achondrogenesis type II (ACG2) is the most severe disorder from amniotic cells of the second and third fetuses revealed that can be produced by dominant mutations in COL2A1.We heterozygosity for a 10370G > T missense mutation (G346V) report on four pregnancies of an apparently healthy, in the COL2A1 gene. This mutation was also found in the nonconsanguineous young couple. The father had scoliosis father, as a mosaic. The couple had a fourth pregnancy, and as a child, and has slight body disproportion with short at 11 weeks fetal hydrops with a septated cystic hygroma trunk. The first child was born at 32 weeks and died were obvious. DNA from CVS demonstrated the same neonatally. In the second pregnancy, short limbs and fetal COL2A1 mutation. ß 2007 Wiley-Liss, Inc. hygroma were noted on ultrasound at 17 weeks’ gestation. Similar findings were observed in the third fetus. Clinical, radiological, and histological evaluation of the fetuses after termination of the pregnancies showed findings consistent Key words: achondrogenesis type II; COL2A1 mutation; with ACG2. Molecular analysis of genomic DNA extracted lethal skeletal dysplasia; mosaicism; recurrence

How to cite this article: Forzano F, Lituania M, Viassolo V, Superti-Furga A, Wildhardt G, Zabel B, Faravelli F. 2007. A familial case of achondrogenesis type II caused by a dominant COL2A1 mutation and ‘‘patchy’’ expression in the mosaic father. Am J Med Genet Part A 143A:2815–2820.

INTRODUCTION members as well. In rare cases, mutations that have resulted in in children have been Achondrogenesis type II (ACG2) is a lethal skeletal found in a mosaic parent who either had Stickler dysplasia which is the most severe of the wide syndrome or mild spondyloepiphyseal dysplasia spectrum of disorders that can be produced by [Winterpacht et al., 1993; Spranger et al., 1994]. In mutations in COL2A1, the gene coding for cartilage- another family, recurrence of ACG2 in two fetuses specific type 2. Mutations within the from the same clinically normal parents was inter- COL2A1 gene also cause preted as evidence for gonadal mosaicism [Faivre (OMIM 200610), Spondyloepiphyseal dysplasia et al., 2004]. (SED) congenita (OMIM 183900), SED Namaqualand Here, we report on a family in which three fetuses type (OMIM 142670), mild SED with precocious were affected by ACG2, and the father, who carries osteoarthritis, spondyloepimetaphyseal dysplasia the mutation in a mosaic state, shows an intermediate Strudwick type (OMIM 184250), Kniest dysplasia phenotype. (OMIM 156550), multiple epiphyseal dysplasia with myopia and conductive deafness, spondyloperiph- eral dysplasia (OMIM 271700), and Stickler dysplasia type I (OMIM 108300). The mutations responsible for *Correspondence to: Dr. F. Forzano, M.D., S.C.Genetica Umana, these phenotypes generally arise de novo—apart E.O.Ospedali Galliera, Via Volta 8, 16128 Genova, Italy. from type I, which is a milder E-mail: [email protected] condition and is frequently identified in family DOI 10.1002/ajmg.a.32047 American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a

2816 FORZANO ET AL. CLINICAL REPORT ultrasonography (ATL 3000, 5 MHz transducer), a viable fetus was recognized with circumferences of The couple first came to our attention after the first the head and abdomen appropriate for gestational child died soon after birth, and the second pregnancy age. All long bones had been measured in all was terminated. A COL2A1-related disease was extremities and showed that the limb shortening suspected based on X-rays and postmortem exami- involved all segments: femur length 39 mm (<1st nations. Both parents were of normal height, but centile; range 49.8–60.1 mm); tibia 32 mm (<3rd the father had scoliosis and showed slight body centile; range 44.4–54.0 mm); fibula 31.7 mm (<3%; disproportion (see description below). Based on range 43.1–52.9 mm); humerus 36.8 mm (<3rd these findings, counseling included the potential centile; range 45.5–55.0 mm); radius 31.9 mm for a recessive phenocopy. Somatic mosaicism was (<3rd centile; range 37.8–47.3 mm); ulna 36.4 mm also considered, although deemed less likely. The (<3rd centile; range 43.2–53.1 mm). The foot length couple had two additional pregnancies, which were was 48 mm (<3rd centile; range 51.4–64.3 mm) and terminated. the femur length/foot length ratio was 0.81 (<3rd The father of this family is 36 years old, and in centile). generally good health. He wore an orthopedic corset Multiple anomalies included: micrognathia, fetal as a child for scoliosis, and a ‘‘ of the skin redundance in the neck and thorax region, and a spine’’ was suspected at that time, but no further ‘‘bell-shaped’’ thorax. The spine had an ovoid shape examinations were performed. and flattening of vertebral bodies. This pregnancy He is now 165 cm tall and shows slight body ended with preterm labor at 32-week gestation and disproportion with long limbs and short neck delivery of a 1,700 g male who died in the newborn and trunk. X-rays (Fig. 1) show normal femoral period. There was a short, thick neck, flat face with a heads, pelvis and lumbar vertebrae, and some deep nasal bridge, micrognathia, and short limbs. thoracic vertebrae show mild flattening and anterior Measurements were: total length 33 cm; vertex- wedging. sacrum 23 cm; upper limb: humerus 6 cm, forearm The first pregnancy of the couple (maternal age 6 cm, hand 4.5 cm; lower limb: femur 6.3 cm, lower 30 years) was evaluated at 28 weeks and 5 days leg 7 cm, foot 5 cm. Radiographs showed a bell- for suspected abnormal development of fetal limbs shaped thorax, platispondily, and wide femural and and mild polyhydramnios. With transabdominal humeral metaphyses (Fig. 2). Autopsy identified a

FIG.1. Radiographs of father’s spine. Note mild flattening and anterior wedging of some thoracic vertebrae and normal femoral heads, pelvis and lumbar vertebrae.

FIG.2. Ultrasound scan and postnatal radiographs of fetus 1. Note redundant skin (arrows on the left), platispondily (arrows on the right), bell-shaped thorax, wide femural and homeral metaphises. American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a

ACHONDROGENESIS TYPE II 2817

FIG.3. Ultrasound scan of fetus 2. Note large and septated nuchal cystic hygroma (arrows; longitudinal and axial scans) and short femur. patent ductus arteriosus, foramen ovale, and dilated 18 mm (<3rd centile; range 19.6–27.2 mm). The left ureter. measurements of the other long bones were between The same 32-year-old mother was referred at the 5th and the 10th centile. Micrognathia was also 16 weeks of pregnancy for fetal karyotyping (amnio- present. centesis), which was 46,XX. At ultrasonography, the The pregnancy was terminated at 18 weeks fetus showed a large and septated nuchal cystic gestation. Postmortem examination showed a hygroma associated with hydrops and hyperechoic female fetus affected by a marked rhizomelic bowel (Fig. 3). Fetal biometry of the head was shortening of limbs. There was a prominent fore- appropriate for gestational age. All long bones were head, frontal bossing, and depressed nasal bridge. below the 3rd centile (micromelia): femur length Measurements were: cranial circumference (CC) 13.5 mm (range 16.5–24.0 mm); tibia 10.3 mm (range 17 cm; thorax circumference (TC) 14 cm; abdominal 13.3–20.5 mm); fibula 10 mm (range 12.8–20.0 mm); circumference (AC) 15 cm; upper limb: humerus humerus 13.5 mm (range 16.7–24.2 mm); radius 3 cm, forearm 3 cm, hand 2.4 cm; lower limb: femur 9.9 mm (range 13.0–20.3 mm); and ulna 10.4 mm 2.5 cm, foot 2.4 cm. Histologic examination of the (range 14.5–22.0 mm). Pregnancy was terminated at knee growth plates showed reduced and disorgan- 17 weeks gestation. ized columnar chondrocytes. Scans are shown in The couple’s third pregnancy (maternal age Figure 4. 33 years) underwent fetal karyotyping at The couple’s fourth pregnancy was screened at 17 þ 3/7 weeks. The circumferences of the head 10.6 weeks for prenatal diagnosis by chorionic villus and abdomen were appropriate for gestational age. sampling. Transvaginal ultrasonography showed a All long bones showed that only the proximal septated nuchal cystic hygroma with enlarged segments were shortened: femur length 20.4 mm jugular lymphatic sacs (see Fig. 5), and generalized (<3rd centile; range 19.4–27.2 mm) and humerus fetal hydrops developed 1 week later. Amniotic fluid,

FIG.4. Ultrasound scan of fetus 3. Note micrognathia (arrow) and short femur. American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a

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FIG.5. Ultrasound transvaginal scan of fetus 4. Note increased nuchal translucency (left, longitudinal scan) and septated nuchal cystic hygroma (right, axial scan). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.] fetal biometry (CRL 45 mm), and heart rate were MATERIALS AND METHODS normal. Pregnancy was terminated at 11 weeks gestation and analysis of CV DNA was performed Ultrasonography has been performed with ATL for COL2A1 mutations. 3000, 5-MHz transducer and with Acuson Sequoia—

FIG.6. DNA sequence analysis of exon 23 of the COL2A1 gene in different tissues. The same GGT ! GTT mutation is shown in all the tissues examinated. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.] American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a

ACHONDROGENESIS TYPE II 2819 Siemens (Mountain View, CA), 6–10 MHz in all No alterations were found in the mother, while the pregnancies. father’s blood DNA harbored a low mutation signal As an index case, the DNA of fetus 2 was screened consistent with mosaicism. In order to better define for mutations in the COL2A1 gene. Exons 21–49 the degree of mosaicism, we performed DNA were amplified by polymerase chain reaction (PCR) analyses on fibroblasts, hair roots, buccal smear, and sequenced. Further analysis of other family and urine. Comparison of the G/T peaks at the members was performed by the amplification of mutation site revealed weak mosaicism in all tissue exon 23 by PCR and direct sequencing. Exons 21–24 samples (Fig. 6 and Table I). In hair root samples 2, 9, were amplified using the forward primer 50-CTG AAA 12, and 13, the ratio of G/T was approximately 50%, CAG TTG CCA AGG CTA C-30 and the reverse primer whereas hair root samples 1, 5, 6, and 14 only 50-TCA CAG TAC TTC AGG CCT CCC-30. Sequence harbored the wild type allele. analyses of exon 23 were carried out with the forward primer 50-CAC AGC TGC TCC CCA GAA DISCUSSION ATT G-30 and the reverse primer 50-ATC CTG GCA GTG CAG GGC TG-30. Fibroblasts were obtained by ACG2 is a lethal skeletal dysplasia characterized by skin biopsy and cultured with a Hamm’s f10, bovine severe micromelia, relatively large head, small fetal serum 25%, glycerol medium. thorax, and protuberant abdomen. Radiologically, Hair roots from the father were taken from different incomplete ossification of vertebral bodies and skull parts of his body, in particular: head (front and can be observed in association with shortening of occiput), thorax, right shoulder, upper limb (fore- the tubular bones and hypoplastic pelvis. Almost arm, wrist, and hand right and left), lower limb (knee, all screened cases have de novo mutations in the leg right and left), and pubis. We also extracted DNA COL2A1 gene. Faivre et al. [2004] first reported a from buccal smears and urine samples. Sperm recurrence of the disease within a family, but no collection was refused by the patient. mutation was found in the parents. This suggested gonadal mosaicism in one of the parents. We describe the first case of proven somatic RESULTS mosaicism for a COL2A1 mutation that led to a lethal We first screened exons 21–49 of COL2A1 in fetus skeletal dysplasia in four offspring who inherited the 2. Molecular analysis revealed a heterozygous mutation. COL2A1 point mutation in exon 23 (G1037T). The Germline or somatic mosaicism is not per se an mutation alters codon 346 (GGT ! GTT) and results exceptional finding in autosomal dominant skeletal in the aminoacid substitution of glycine to valine dysplasias, since it has been previously documented (G346V). for FGFR3 [, Fryns et al., 1983], COMP

TABLE I. DNA Sequence Analyses of Blood, Fibroblast, Buccal Smear and Hair Root Samples. Mosaicism is Present in All Cell Lines, Although at Different Levels

Nucleotide in position RPA (result file peak area) c.1037 exon 23, gene COL2A1 TG

1235-04 blood G/T 0.076 0.581 1235-04 blood ek 1 G/T 0.182 0.924 1235-04 blood ek 2 G/T 0.171 0.930 1235-04 blood ek 3 G/T 0.152 0.858 1235-04 blood ek 4 G/T 0.152 0.932 1420-05 fibroblasts G/T 0.267 0.744 1378-05 buccle smear G/T 0.174 0.735 1377-05 urine G/T 0.164 0.834 1280-05 hair roots (1) G/T 0.247 0.712 1280-05 hair roots (2) G/T 0.295 0.668 1280-05 hair roots (3) G 0.001 0.997 1280-05 hair roots (4) G 0.062 0.993 1280-05 hair roots (5) G/T 0.229 0.793 1280-05 hair roots (6) G/T 0.164 0.862 1280-05 hair roots (7) G 0.020 0.898 1280-05 hair roots (8) G 0.010 1.000 1280-05 hair roots (9) G/T 0.356 0.649 1280-05 hair roots (10) G 0.023 0.928 1280-05 hair roots (11) G 0.000 1.034 1280-05 hair roots (12) G/T 0.370 0.583 1280-05 hair roots (13) G/T 0.415 0.533 1280-05 hair roots (14) G/T 0.248 0.674 American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a

2820 FORZANO ET AL. [, Ferguson et al., 1997], and Dr. Mauro Castagnetta from Galliera Genetic Bank COL1A1 [, Cohn et al., 1990] (Galliera Hospital, Genova), which is supported mutations. When somatic mosaicism was identified by Telethon Italia, and Mrs. Carmen Marciano in a parent, the manifestations, if any, ranged from for helping with the arrangement and delivery of minimal to severe phenotypes. Different phenotypes samples. Dr. Francesca Madia provided kits for DNA also include mild Stickler syndrome and SED in extraction from urines and buccal smears. This article parents with mosaic COL2A1 mutations who con- is dedicated to the memory of Professor Gianni ceived children affected by a severe Kniest dysplasia Camera, who first proposed a COL2A1 mutation in a [Spranger et al., 1994]. mosaic state in the father in this family. The father of the family in this report has a regional, ‘‘patchy’’ expression of COL2A1-related abnormal- ities, with some bones affected while others are REFERENCES entirely unaffected. This suggests that somatic mosaicism can lead to a Cohn DH, Starman BJ, Blumberg B, Byers PH. 1990. Recurrence of lethal osteogenesis imperfecta due to parental mosaicism milder but generalized clinical phenotype, which for a dominant mutation in a human gene might be determined by the time of onset and the (COLIAI). Am J Hum Genet 46:591–601. body region or segment in which the somatic muta- Faivre L, Le Merrer M, Douvier S, Laurent N, Thauvin-Robinet C, tion arises. Rousseau T, Vereecke I, Sagot P, Delezoide AL, Coucke P, Mortier G. 2004. Recurrence of achondrogenesis type II within Finally, this case of COL2A1 mosaicism reinforces the same family: Evidence for germline mosaicism. Am J Med the need to consider mosaicism when counseling Genet Part A 126A:308–312. families with newly recognized COL2A1-related Ferguson HL, Deere M, Evans R, Rotta J, Hall JG, Hecht JT. 1997. disorders. We recommend diagnostic prenatal ultra- Mosaicism in pseudoachondroplasia. Am J Med Genet 70: sound screening as follow-up for subsequent preg- 287–291. Fryns JP, Kleczkowska A, Verresen H, Van Den Berghe H. 1983. nancies, especially when a gene mutation is not Germline mosaicism in achondroplasia: A family with three identified or when the parents refuse invasive exami- affected siblings of normal parents. Clin Genet 24:156–158. nations, Recurrence of the disease can be identified Soothill PW, Vuthiwong C, Rees H. 1993. Achondrogenesis type 2 by ultrasound at the earliest around the 12th week of diagnosed by transvaginal ultrasound at 12 weeks’ gestation. Prenat Diagn 13:523–528. gestation [Soothill et al., 1993], and include polyhy- Spranger J, Menger H, Mundlos S, Winterpacht A, Zabel B. 1994. dramnios, nuchal edema, and shortening of limbs. Kniest dysplasia is caused by dominant collagen II (COL2A1) mutations: Parental somatic mosaicism manifesting as Stickler phenotype and mild spondyloepiphyseal dysplasia. Pediatr ACKNOWLEDGMENTS Radiol 24:431–435. Winterpacht A, Hilbert M, Schwartze U, Mundlos S, Spranger J, We thank the family for prompt collaboration Zabel B. 1993. Kniest and Stickler dysplasia phenotypes and perseverance they always demonstrated. We caused by collagen type II gene (COL2A1) defect. Nat Genet also thank Dr. Chiara Baldo, Dr. Massimo Mogni, 3:323 326.